Go is one of a family of the guanine nucleotide-binding proteins (G proteins) that are important mediators of signal transduction across biological membranes. Although the precise function of Go is unclear, it appears to be closely associated with calcium flux and receptor-mediated events. Like the other G proteins, Go is a heterotrimer of alpha, beta, and gamma subunits. A cDNA clone for the alpha subunit of Go was previously isolated and sequenced in this laboratory, and has been used for expression in E. coli and in COS cells. E. coli expressed the Go alpha cDNA cloned into the vector pRC-23, with the resulting recombinant protein (rGo alpha) being produced at high levels (1- 2% of cellular protein). rGo alpha was immunoreactive with polyclonal antisera raised against bovine brain Go alpha and served as substrate for pertussis toxin-catalyzed ADP-ribosylation. The ADP-ribosylation, like that of bovine brain Go alpha, was enhanced by the presence of beta and gamma subunits. Furthermore, ADP- ribosylation of rGo alpha was stimulated by GDP and GTP, but inhibited by non-hydrolyzable GTP analogs, indicating that rGo alpha contains a functional guanine nucleotide binding site. Single point mutations were carried out in the cDNA of Go alpha and two mutants have been expressed in E. coli: 1) with cysteine 351 (the site of pertussis toxin catalyzed ADP-ribosylation) replaced by glycine; 2) with glycine 352 replaced by aspartic acid. These recombinant mutant proteins reacted with anti-Go alpha polyclonal antiserum, but they did not serve as substrates for pertussis toxin-catalyzed ADP-ribosylation.